In a competitive immunoassay, external reagent that competes with the analyte has to be added, which is not the case in the non-competitive assay format. The method of choice for the detection of analytes by immuno-chemistry is nowadays a non-competitive immunoassay, where two antibodies bind to two different epitopes of the analyte creating a so-called sandwich-type assay. Such an assay is well suited for high molecular weight analytes and it provides improved speed, sensitivity, and specificity, which are needed in modern immunoassays. However, it has been a difficult task to develop non-competitive assays for small analytes, because low molecular weight molecules are not large enough for binding simultaneously to more than one antibody independently. Therefore, despite many fundamental problems with respect to specificity and sensitivity, the competitive immunoassay format has been almost exclusively used for the detection of small analytes.
However, there are few publications where the development of a non-competitive immunoassay for a small analyte has been reported. In these papers, a secondary anti-immune complex (anti-IC) antibody, which binds primary anti-analyte antibody that is combined with the analyte but which does not bind the primary antibody or the analyte alone, has been developed (Ullman et al., 1993; Self et al., 1994; Towbin et al., 1995). Ullman et al., 1993, describe an antibody that recognizes an immune complex of an antibody to tetrahydrocannabinol (THC). The anti-IC antibody was obtained by using an affinity labelled anti-THC antibody as immunogen and selecting an anti-IC antibody the binding of which was enhanced by the presence of Δ9THC. Self et al., 1994, used the same principle in preparing anti-IC antibodies for determining digoxin. Towbin et al., 1995, report a sandwich immunoassay for the hapten angiotensin II, wherein the immunisation involves tolerization with uncomplexed primary antibody prior to immunisation with the anti-immune complex to obtain the anti-IC antibodies. The anti-IC antibodies used in non-competitive immunoassays for small analytes have so far been conventional polyclonal or monoclonal antibodies obtained by immunisation, and the assays described here include labelling of the primary antibody and immobilisation of the secondary or vice versa.
So-called ‘idiometric’ non-competitive immunoassay for small analytes has been developed by Mares et al., 1995. They used two types of anti-idiotypic antibodies, which recognize different epitopes within the hypervariable region of the oeastradiol specific primary antibody. The first anti-idiotypic antibody (betatype) possesses the capacity of competing with the analyte for an epitope at the binding site of the primary antibody. The second anti-idiotype (alphatype) recognizes an epitope within the variable region of the primary antibody and the binding is not sensitive to the presence of the analyte. The alphatype is, however, sterically hindered from binding to the primary antibody in the presence of the betatype. These three types of antibodies permit the development of a non-competitive assay for small analytes.
So-called open sandwich immunoassays have been developed for the detection of haptens (Suzuki et al., 2000; Suzuki et al., 1999; Yokozeki et al., 2002). They are non-competitive assays based on a phenomenon according to which the association of separated VH and VL chains in some antibodies is strongly favoured in the presence of antigen (Ueda et al., 1996).
Despite the significant benefits of the non-competitive immunoassay format, only few examples of that kind of assays for small analytes have been reported. The reason for that is most probably the difficulty of producing secondary (anti-immuno complex, or anti-idiotypic) antibodies by immunising animals. For example anti-hapten monoclonal antibodies, which have been developed by hybridoma technology (Kohler and Milstein, 1975), are self-antigens for mice and raising immunoresponse against them is difficult (Maruyama et al., 2002; Ullman et al., 1993; Kobayashi et al., 2000). A further problem is that the immune complex used for immunisation tends to break down before the response to the immuno complex is obtained (Ullman et al., 1993; Kobayashi et al., 2000).
The present invention now provides a non-competitive immunoassay protocol for small analytes, which circumvents the immunisation of animals with the immune complex, which has been so far the most challenging task when anti-IC antibodies have been developed. The invention also facilitates a homogenous immunoassay, which further improves the speed, sensitivity and simplicity of the assay.